In the yeast Saccharomyces cerevisiae, CTP, the CTP synthetase reaction product, is a precursor of phospholipids that are synthesized by the CDP-diacylglycerol and Kennedy (CDP-choline) pathways as well as all membrane phospholipids. Studies performed during the current grant period showed that CTP synthetase activity plays a regulatory role in the utilization of the pathways used for the synthesis of phosphatidylcholine. CTP synthetase was purified, characterized, and shown to be activated by phosphorylation via protein kinase A and protein kinase C and inhibited by CTP. In this competitive continuation grant application we propose molecular genetic and biochemical studies that are expected to provide insight into the regulation of CTP synthetase activity by phosphorylation and by CTP product inhibition and into the physiological relevance of this regulation with respect to phospholipid synthesis. They propose to examine the hypothesis that the regulation of CTP synthetase activity by phosphorylation involves the nucleotide-dependent tetramerization of the enzyme. CTP synthetase mutants will be isolated with altered protein kinase A and protein kinase C phosphorylation sites. These mutants will be used for physiological and biochemical studies to examine the effects of phosphorylation on the regulation of CTP synthetase activity. The effects of this regulation of phospholipid synthesis will be propose to isolate mutants that are defective in a proposed CTP binding site in CTP synthetase. These mutants will be used for physiological and biuochemical studies to examine the regulation of CTP synthetase activity by CTP product inhibition. The effect of this regulation on phospholipid synthesis will be examined. The results from the proposed studies should be relevant to higher eukaryotic organisms and be of great interest to, and complement the work of, other investigators in the field of phospholipid metabolism. This work should also be relevant to other aspects of macromolecular synthesis that are dependent on CTP as a precursor.